Eukaryotic cells reversibly assemble the actin protein into filaments which are then arrayed by accessory proteins into the actin cytoskeleton (AC). Disruption of the AC is associated with significant cell softening and in the case of migrating cells it also results in loss of motility. The exact role of the AC in the mechanical strength of cells is not fully understood. The AC can be roughly divided into two superimposing elastic elements: a homogeneous network of protein filaments predominantly underlying the plasma membrane and stress fibers which span the cell interior. This proposal will focus on the homogeneous cortical part of the AC. The proposed research takes two novel approaches: (1) in vitro measurements will investigate how the elastic response of actin networks is generated on the microscopic level of a single actin filament in a network and (2) in vivo measurements of the specific elasticity of the cortical AC will determine the role of actin networks for whole cell elasticity. Simultaneous measurements of the elastic properties of F-actin solutions/gels and fluorescence microscopy of individual constituent actin filaments under shear will provide detailed characterization of the interactions between actin filaments which cause the elastic behavior. These data will distinguish between recently suggested models and lead to quantitative predictions of the strength of the AC as a function of its composition. This knowledge will improve the understanding of cell motility. A particular focus will be on how transient crosslinkers and molecular motors modulate the strength of these networks. Preliminary results indicate that inactive myosin II acts as a crosslinker, whereas active myosin liquefies F-actin networks in the absence of other crosslinking proteins. The dependence of the mechanical strength on filament stiffness will be investigated as an additional possibility in modulating the strength of actin networks. To accurately measure cell elasticity of large, statistically significant numbers of cells, an optical tool has been developed to non- destructively stretch single cells between two beams of a laser. Measurements of cells in suspension, which show a peripheral AC but no stress fibers, will allow the PI to quantify the contribution of actin networks to cell elasticity. The contribution of these networks to the overall mechanical strength of cells will be determined by measuring the elasticity of cells with varying ACs. This also allows determination of the relevance of in vitro research on homogeneous actin networks for the cell cytoskeleton. A primary focus will be the extent to which malignant transformation of cells by oncogenic vectors -which is correlated with changes of the AC - can be monitored by cell elasticity measurements.